Search

Background: Phosphorylation of the intracellular domain of the EPHA2 receptor tyrosine kinase (RTK) on serine 897 (S897) has been demonstrated to mediate EPHA2 oncogenic activity. Here, we show that in thyroid cancer cells harboring driver oncogenes that signal through the extracellular regulated kinase (ERK1/2) signaling pathway [rearranged RET RTK (RET/PTC), KRAS(G12R), or BRAF V600E oncogenes], EPHA2 is robustly phosphorylated on S897. EPHA2 S897 is embedded in a consensus sequence for phosphorylation by the AGC family kinases, including p90RSK (ribosomal protein S6 kinase), a direct ERK1/2 target. Methods: We show that recombinant p90RSK phosphorylates in vitro EPHA2 S897 and that treatment with chemical inhibitors targeting p90RSK or other components of the ERK1/2 pathway blunts S897 phosphorylation. Results: RNA interference-mediated knockdown combined with rescue experiments demonstrated that EPHA2 S897 phosphorylation mediates thyroid cancer cell proliferation and motility. Conclusions: These findings point to EPHA2 S897 as a crucial mediator of the oncogenic activity of the ERK1/2 signaling cascade in thyroid cancer.

Sonic Hedgehog (Shh) pathway regulates embryonic development of different organs including the thyroid gland. The aberrant activation of Shh signaling has been found in several types of cancer and according to recent evidences it represents an important regulator of tumor-stroma interaction. In this study, we have analyzed expression, activation and molecular mechanisms regulating the Shh pathway and its involvement in the modulation of tumor stroma interaction in anaplastic thyroid cancer (ATC) cells. Our results suggest that Shh signaling undergoes a dual mechanism of induction in ATC cells: 1) a basal non-canonical Smo-dependent activation of Gli transcription factor that is partly caused by interaction with the RAS/BRAF/MEK oncogenic pathway and is characterized by the absence of Shh ligand expression in thyroid cancer cells and 2) a paracrine response of cancer cells to Shh ligand secreted by tumor stroma (fibroblasts and mesenchymal stromal cells, MSCs) inducing cancer cell migration and in vitro tumorigenesis. Our data therefore suggest Shh as a potential novel therapeutic target in aggressive thyroid cancers., Competing Interests: CONFLICTS OF INTEREST None.

NCOA4 is a transcriptional coactivator of nuclear hormone receptors that undergoes gene rearrangement in human cancer. By combining studies in Xenopus laevis egg extracts and mouse embryonic fibroblasts (MEFs), we show here that NCOA4 is a minichromosome maintenance 7 (MCM7)-interacting protein that is able to control DNA replication. Depletion-reconstitution experiments in Xenopus laevis egg extracts indicate that NCOA4 acts as an inhibitor of DNA replication origin activation by regulating CMG (CDC45/MCM2-7/GINS) helicase. NCOA4(-/-) MEFs display unscheduled origin activation and reduced interorigin distance; this results in replication stress, as shown by the presence of fork stalling, reduction of fork speed, and premature senescence. Together, our findings indicate that NCOA4 acts as a regulator of DNA replication origins that helps prevent inappropriate DNA synthesis and replication stress., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Here, we show that the anaplastic thyroid carcinoma (ATC) features the up-regulation of a set of genes involved in the control of cell cycle progression and chromosome segregation. This phenotype differentiates ATC from normal tissue and from well-differentiated papillary thyroid carcinoma. Transcriptional promoters of the ATC up-regulated genes are characterized by a modular organization featuring binding sites for E2F and NF-Y transcription factors and cell cycle-dependent element (CDE)/cell cycle gene homology region (CHR) cis-regulatory elements. Two protein kinases involved in cell cycle regulation, namely, Polo-like kinase 1 (PLK1) and T cell tyrosine kinase (TTK), are part of the gene set that is up-regulated in ATC. Adoptive overexpression of p53, p21 (CIP1/WAF1), and E2F4 down-regulated transcription from the PLK1 and TTK promoters in ATC cells, suggesting that these genes might be under the negative control of tumor suppressors of the p53 and pRB families. ATC, but not normal thyroid, cells depended on PLK1 for survival. RNAi-mediated PLK1 knockdown caused cell cycle arrest associated with 4N DNA content and massive mitotic cell death. Thus, thyroid cell anaplastic transformation is accompanied by the overexpression of a cell proliferation/genetic instability-related gene cluster that includes PLK1 kinase, which is a potential molecular target for ATC treatment.

RAI, also named ShcC/N-Shc, one of the members of the Shc proteins family, is a substrate of the RET receptor tyrosine kinase. Here, we show that RAI forms a protein complex with both RET/MEN 2 A and RET/PTC oncoproteins. By co-immunoprecipitation, we found that RAI associates with the Grb 2-associated binder 1 (GAB 1) adapter. This association is constitutive, but, in the presence of RET oncoproteins, both RAI and GAB 1 are tyrosine-phosphorylated, and the stoichiometry of this interaction remarkably increases. Consequently, the p 85 regulatory subunit of phosphatidylinositol-3 kinase (PI-3 K) is recruited to the complex, and its downstream effector Akt is activated. We show that human thyroid cancer cell lines derived from papillary or medullary thyroid carcinoma (PTC or MTC) carrying, respectively, RET/PTC and RET/MEN 2 A oncoproteins express RAI proteins. We also show that human PTC samples express higher levels of RAI, when compared to normal thyroid tissue. In thyroid cells expressing RET/PTC 1, ectopic expression of RAI protects cells from apoptosis; on the other hand, the silencing of endogenous RAI by small inhibitory duplex RNAs in a PTC cell line that expresses endogenous RET/PTC 1, increases the rate of spontaneous apoptosis. These data suggest that RAI is a critical substrate for RET oncoproteins in thyroid carcinomas., (Oncogene (2005) 24, 6303-6313.)

In papillary thyroid carcinomas (PTCs), rearrangements of the RET receptor (RET/PTC) and activating mutations in the BRAF or RAS oncogenes are mutually exclusive. Here we show that the 3 proteins function along a linear oncogenic signaling cascade in which RET/PTC induces RAS-dependent BRAF activation and RAS- and BRAF-dependent ERK activation. Adoptive activation of the RET/PTC-RAS-BRAF axis induced cell proliferation and Matrigel invasion of thyroid follicular cells. Gene expression profiling revealed that the 3 oncogenes activate a common transcriptional program in thyroid cells that includes upregulation of the CXCL1 and CXCL10 chemokines, which in turn stimulate proliferation and invasion. Thus, motile and mitogenic properties are intrinsic to transformed thyroid cells and are governed by an epistatic oncogenic signaling cascade.

Activating germ-line point mutations in the RET receptor are responsible for multiple endocrine neoplasia type 2-associated medullary thyroid carcinoma (MTC), whereas somatic RET rearrangements are prevalent in papillary thyroid carcinomas (PTCs). Some rare kindreds, carrying point mutations in RET, are affected by both cancer types, suggesting that, under specific circumstances, point mutations in RET can drive the generation of PTC. Here we describe a family whose siblings, affected by both PTC and MTC, carried a germ-line point mutation in the RET extracellular domain, converting cysteine 634 into serine. We tested on thyroid follicular cells the transforming activity of RET(C634S), RET(K603Q), another mutant identified in a kindred with both PTC and MTC, RET(C634R) a commonly isolated allele in MEN2A, RET(M918T) responsible for MEN2B and also identified in kindreds with both PTC and MTC, and RET/PTC1 the rearranged oncogene that characterizes bona fide PTC in patients without MTC. We show that the various RET point mutants, but not wild-type RET, scored constitutive kinase activity and exerted mitogenic effects for thyroid PC Cl 3 cells, albeit at significantly lower levels compared to RET/PTC1. The low mitogenic activity of RET point mutants paralleled their reduced kinase activity compared to RET/PTC. Furthermore, RET point mutants maintained a protein domain, the intracellular juxtamembrane domain, that exerted negative effects on the mitogenic activity. In conclusion, RET point mutants can behave as dominant oncogenes for thyroid follicular cells. Their transforming activity, however, is rather modest, providing a possible explanation for the rare association of MTC with PTC.

Papillary thyroid carcinomas are characterized by rearrangements of the RET receptor tyrosine kinase generating RET/PTC oncogenes. Here we show that osteopontin (OPN), a secreted glycoprotein, is a major RET/PTC-induced transcriptional target in PC Cl 3 thyroid follicular cells. OPN upregulation depended on the integrity of the RET/PTC kinase and tyrosines Y1015 and Y1062, two major RET/PTC autophosphorylation sites. RET/PTC also induced a strong overexpression of CD44, a cell surface signalling receptor for OPN. Upregulation of CD44 was dependent on RET/PTC Y1062, as well. Constitutive OPN overexpression or treatment with exogenous recombinant OPN sharply increased proliferation, Matrigel invasion and spreading in collagen gels of RET/PTC-transformed PC Cl 3 cells. These effects were impaired by the treatment of PC Cl 3-RET/PTC cells with OPN- and CD44-locking antibodies. Thus, RET/PTC signalling triggers an autocrine loop involving OPN and CD44 that sustains proliferation and invasion of transfomed PC Cl 3 thyrocytes.

Noninvasive thyroid nodules that exhibit borderline morphological signs of papillary cancer are difficult to diagnose and we do not know if they represent papillary carcinoma precursor lesions. Forty-six such nodules were analyzed for RET activation by immunohistochemistry and, in selected cases, by reverse transcriptase-polymerase chain reaction performed on RNA extracted after laser capture microdissection (LCM) of the tumor foci with and without papillary carcinoma features and positive RET immunoreactivity. RET immunoreactivity was identified, at least focally, in 30 of 46 (65.2%) of the nodules where it closely paralleled the morphological changes. Enough RNA was obtained after LCM in seven samples. RET/PTC1 or RET/PTC3 were detected in microscopic foci with papillary carcinoma features in most of the thyroid nodules (five of seven cases). No RET/PTC1 or RET/PTC3 rearrangements were detected in areas of the same tumors that lacked the cytological alterations. Analysis of clonality in the same nodules selected for LCM demonstrated that two were monoclonal and six were polyclonal. We conclude that RET activation closely parallels the morphological changes, that it is restricted to those areas of the tumor with the cytological alterations and that it is detectable in both mono- and polyclonal tumors. Although the finding of microscopic foci indicative of papillary carcinoma in a hyperplastic or adenomatous nodule does not justify the interpretation of the entire lesion as papillary carcinoma, it is possible that such foci may precede the development of invasive papillary cancer.

The proto-oncogene RET encodes a transmembrane growth neurotrophic receptor with tyrosine kinase (TK) activity. RET mutations are associated with several human neoplastic and nonneoplastic diseases, including thyroid papillary carcinoma, multiple endocrine neoplasia type 2 syndromes, and Hirschsprung's disease. Activation of receptor TKs results in the binding and activation of downstream signaling proteins, among which are nonreceptor TKs of the Src family. To test the involvement of c-Src in Ret-mediated signaling, we measured the levels of c-Src activity in NIH3T3 cells coexpressing Ret and the accessory GFR alpha-1 receptor or an epidermal growth factor receptor/Ret chimeric receptor when the cells were stimulated by glial cell line-derived neurotrophic factor or epidermal growth factor, respectively. Ret stimulation resulted in the activation of c-Src. We also measured the levels of Src kinase activity in cell lines expressing isoforms of the Ret receptor activated by different mutations. These cells showed higher Src kinase activity than the normal counterpart. Furthermore, we show that Ret is able to associate with the SH2 domain of Src in a phosphotyrosine-dependent fashion. Microinjection of a kinase inactive mutant of c-Src blocked Ret-mediated mitogenic effect. These experiments demonstrate that activated Ret is able to bind and stimulate c-Src kinase and that Src activation is essential for the mitogenic activity of Ret.

The most frequent genetic alterations described thus far in human papillary thyroid carcinomas are somatic rearrangements of the RET proto-oncogene, which generate the chimeric RET/PTC oncogenes. We recently found that the expression of the RET/PTC1 oncogene blocked the expression of the thyroid-differentiated phenotype in rat thyroid epithelial cell line PC CI 3 (PC). Here, we show that this block occurs at a transcriptional level; indeed, the thyroid-specific thyroglobulin and thyroperoxidase gene promoters were inactive in PC-PTC cells. Specific transcription factors, namely, TTF-1 and Pax-8, regulate the expression of differentiated functions in thyroid cells. Here, we show that Pax-8 is expressed at reduced levels in PC-PTC cells and that its adoptive overexpression is unable to restore the activity of target promoters. In contrast, TTF-1 expression is unaltered in PC-PTC cells; however, by using a synthetic promoter that contains its specific target sequence, we demonstrate that TTF-1 is inactive in PC-PTC cells. We conclude that the RET/PTC1 oncogene alters the expression of the thyroid-differentiated phenotype by at least two different mechanisms, ie., down-regulation of Pax-8 protein and mRNA expression and impaired function of TTF-1 and Pax-8, which occurs at a posttranslational level.

Specific point-mutations of the RET receptor tyrosine kinase protooncogene are responsible for the inheritance of multiple endocrine neoplasia type 2A (MEN2A) and 2B (MEN2B), and familial medullary thyroid carcinoma (FMTC). MEN2B is caused by the substitution of methionine 918 by a threonine in the tyrosine kinase (TK) domain of RET. This mutation converts RET into a dominant transforming oncogene. We have substituted Met918 with four different residues and found that RET acquired transforming activity only when Met918 was substituted with a threonine. However, also when serine and valine, but not leucine or phenylalanine, were inserted in position 918, the RET TK function was activated and induced, especially in the case of the RET(918Ser), immmediate-early response genes. We conclude that the preservation of Met918 is critical for the control of RET kinase. However, only when a threonine residue is present in position 918, does RET efficiently couple with a transforming pathway.

Distinct point mutations of RET, a tyrosine-kinase receptor encoding gene, are responsible for the inheritance of multiple endocrine neoplasia type 2 syndromes (MEN2A and MEN2B) and familial medullary thyroid carcinoma (FMTC). In particular, MEN2A is a more complex and aggressive disease than FMTC, being characterized by pheochromocytomas and parathyroid alterations, in addition to medullary thyroid carcinomas. The mutations associated with MEN2A and FMTC affect one of five cysteine residues mapping in the extracellular domain of the Ret protein. However, recent studies have indicated that MEN2A and FMTC disease phenotypes correlate with the position of mutations in RET. Mutations of Cys-634 are more frequent in families with MEN2A, whereas Cys-620 mutations are very rarely found in MEN2A patients and, in contrast, are frequently found in FMTC patients. We have reported previously that mutations of Cys-634 constitutively activate the RET transforming potential by causing a disulfide bridge-mediated homodimerization. Here, we report that the mutation Cys-620 --> Tyr is able to cause a constitutive dimerization of Ret, with consequent activation of its kinase and transforming activities, to a lower extent than mutation of Cys-634. We suggest that the difference in ability to activate RET shown by mutations associated with FMTC and MEN2A represents the molecular basis of the phenotypic diversity between the two syndromes.

Amplification of erbB-2 gene and overexpression of gp185erbB-2 gene product is found in approximately one-third of primary human breast and ovarian cancer. Overexpression of gp185erbB-2 was recently found in human papillary thyroid carcinomas, but not in thyroid follicular carcinomas or adenomas. The erbB-2 gene encodes a cell surface growth factor receptor with intrinsic tyrosine kinase activity. Wild type human erbB-2 has been shown to act as a potent oncogene when overexpressed in mouse fibroblasts. To test whether overexpression of normal human erbB-2 gene can transform epithelial differentiated rat thyroid cells, these cells were infected with a recombinant retroviral expression vector containing the erbB-2 protooncogene. Rat thyroid cells expressing high levels of gp185erbB-2 do not display a fully transformed and tumorigenic phenotype. However, the isolated cell clones that overexpress gp185erbB-2, show changes in their growth properties if compared to normal thyroid cells, since they can grow in absence of thyrotropin, the main growth factor controlling thyroid cell proliferation in vitro, and do not respond to the growth inhibitory effect of transforming growth factor beta.

Optimal growth and differentiation of normal rat thyroid FRTL5 cells depend strictly on the presence of thyroid-stimulating hormone (TSH). FRTL5 cells deprived of TSH cease dividing and become quiescent. Addition of TSH to quiescent cells, which activates the cyclic AMP-mediated pathway, is sufficient to stimulate cell entry into S phase of the cell cycle. We have previously shown that the differential expression of the two isozymes, type I and type II, of the cyclic AMP-dependent protein kinase (PKA) correlates with cell growth and differentiation of several rodent and human cell lines. We have studied the role of PKA in the TSH-regulated growth and cell cycle distribution of FRTL5 cells. Upon addition of TSH to FRTL5 cells deprived of hormone, a rapid induction of RI alpha mRNA species occurred within 30 min after treatment, reaching the levels of proliferating FRTL5 cells at 12 h. RII alpha mRNA levels slightly increased after TSH addition, whereas C alpha mRNA levels did not show major changes. Photoaffinity labeling of PKA receptor proteins showed that addition of TSH to quiescent FRTL5 cells induced a progressive increase in RI alpha levels starting at 6 h after stimulation, whereas RII alpha receptor levels increased only slightly. When FRTL5 cells were treated with an antisense oligodeoxynucleotide targeted against the RI alpha regulatory subunit, their growth was arrested, whereas an antisense against the RII alpha regulatory subunit produced only a mild growth inhibition. Moreover, exposure to the antisense RI alpha oligomer resulted in accumulation of cells in the G0-G1 compartment, as during TSH deprivation.(ABSTRACT TRUNCATED AT 250 WORDS)

Transforming growth factor beta (TGF beta) is a secreted polypeptide factor that is thought to play a major role in the regulation of proliferation of many cell types and various differentiation processes. TGF beta acts on thyroid cells by inhibiting cell proliferation and expression of differentiation markers, such as thyroglobulin production and iodide uptake. Exponentially growing thyroid cells cultures accumulate mostly in G0/G1 after exposure to TGF beta for 48 hours. TGF beta inhibits the TSH induced transition of quiescent thyroid cell from the G0 to the S phase. These effects on the thyroid cell growth, however, are not mediated by changes in the TSH-induced cell cycle-related genes expression; both immediate early and progression genes expression is unaffected by the TGF beta treatment.

Rat thyroid cells in culture (FRTL-5 strain) require thyrotropic hormone (TSH) for growth. TSH alone in serum free medium is able to induce DNA synthesis of FRTL-5 cells. DNA synthesis occurs 18-20 hours following TSH stimulation of quiescent cells. Here we demonstrate that two sets of genes, related to the entry of cells in the S phase, are induced by TSH: 1) immediate early genes, such as c-jun and a gene coding for a zinc-finger protein Xrox 20/Egr2, both having a pattern of expression similar to the c-fos oncogene; 2) early delayed genes such as ornithine decarboxylase (ODC), 2F-1, a gene that shows a strong similarity in aminoacid sequence to a mitochondrial ADP/ATP carrier, and the asparagine synthetase gene (TS11). Furthermore, an increased expression of the histone H3 gene, a typical marker of S phase, has been observed in TSH-treated FRTL-5 cells.

To evaluate the role of transforming growth factor alpha (TGF alpha) in transformed rat thyroid epithelial cells, expression and production of TGF alpha were measured in a normal rat thyroid epithelial cell line, FRTL-5, and in the same cells transformed by the Ki-ras oncogene. FRTL-5 cells transformed with Ki-ras exhibit a 5- to 7-fold increase in the levels of TGF alpha-specific mRNA and a 3- to 4-fold enhancement of the amounts of TGF alpha protein in the conditioned medium, as compared with the normal thyroid cells. Although conditioned medium from the Ki-ras transformed FRTL-5 cells or authentic epidermal growth factor or TGF alpha are able to stimulate the anchorage-dependent growth of nontransformed FRTL-5 cells, neither conditioned medium nor the growth factors are able to induce the anchorage-independent growth of these cells in soft agar. FRTL-5 cells were then transfected with an expression vector plasmid containing the human TGF alpha cDNA to directly ascertain if over-expression of this growth factor is able to induce transformation in these cells. The TGF alpha-transfected FRTL-5 clones constitutively produced high amounts of TGF alpha at a level equivalent to or greater than the level found in the conditioned medium from the Ki-ras transformed FRTL-5 clones. Moreover, in contrast to the Ki-ras transformed cells, the TGF alpha transfectants were unable to grow in soft agar, did not form tumors in nude mice, and showed no reduction in the secretion of thyroglobulin. These data demonstrate that unlike Ki-ras, the constitutive expression of biologically active TGF alpha is not entirely sufficient to elicit a transformed phenotype in these cells.

The action of transforming growth factor-beta (TGF-beta) on the morphology, cytoskeleton and extracellular matrix was investigated in FRTL-5 thyroid epithelial cells. After treatment with TGF-beta, FRTL-5 cells became flat and developed straight and thick bundles of actin microfilaments. This effect of TGF-beta was observed even in the presence of thyrotropin, which has a strong microfilament disrupting action. TGF-beta also influenced some aspects of the extracellular matrix organization. Immunofluorescence staining of FRTL-5 cells revealed both the appearance of a fibrillar array of fibronectin in association with the basal plasma membrane and a change in the morphology of basally located laminin patches. TGF-beta induced the formation of adhesion structures at the ventral portion of the cell membrane. Vinculin was focally concentrated at the end of stress fibers in areas corresponding to focal adhesions as revealed by interference reflection microscopy (IRM). The ability to modulate cytoskeleton organization and extracellular matrix protein distribution might mediate some of the reported TGF-beta effects on the expression of specific functional properties in thyroid cells.

Rat FRTL-5 and PC-Cl-3 thyroid cells are continuously cultured, clonal lines which require thyrotropin to grow and function. Both can be efficiently transformed when infected with RNA or DNA viruses carrying oncogenes or when directly transfected with activated oncogenes. Transformation, assayed by the appearance of cell growth in agar and by tumorigenicity in syngeneic rats or nude mice, is associated with the loss of thyrotropin-dependent cell division and thyrotropin-regulated functions such as thyroglobulin synthesis. In 16 clones of FRTL-5 or PC-Cl-3 cells transformed with different oncogenes, we show that loss of thyrotropin-dependent growth and function correlates with the loss of thyrotropin receptor gene expression, measured with a rat thyrotropin receptor cDNA probe.

The regulation of adenylate cyclase has been analyzed in normal rat thyroid cells as well as in the same cells transformed by the v-ras-k oncogene. In both cell types the adenylate cyclase complex consists of the two GTP-binding proteins, Gi and Gs, as demonstrated by the specific ADP-ribosylation induced by pertussis and cholera toxin, respectively. The response of adenylate cyclase of the transformed cells to forskolin, pertussis toxin and cholera toxin is attenuated with respect to the control cell line. The thyrotropic hormone (TSH), that acts on normal thyroid cells in culture as a growth factor by stimulating the adenylate cyclase activity, is not able to induce DNA synthesis nor does it stimulate adenylate cyclase in v-ras-k transformed cells.

Rat thyroid cells in culture, rendered quiescent by hormone deprivation, can be stimulated to undergo DNA synthesis in the absence of serum by the addition of purified thyrotropin. The primary effect in response to thyrotropin action in thyroid cells is the induction of the c-fos oncogene, followed by c-myc expression. This suggests that thyrotropin acts as a competence growth factor.

Transforming growth factor beta (TGF beta) is now known to have a number of effects other than inducing phenotypic transformation of fibroblastic cells: TGF beta controls proliferation, differentiation, and other functions in many cell types. In this paper we have analyzed the action of TGF beta 1 on a system of differentiated epithelial rat thyroid cells in culture (FRTL5), which depend on the addition of thyrotropin for their growth. TGF beta 1 is able to inhibit the growth of thyroid cells by reducing cell response to thyrotropin. Moreover, in analogy to the effect produced upon other differentiated cells in culture, such as myoblasts and adipocytes, TGF beta 1 modulates the expression of FRTL5-specific thyroid markers, reducing thyroglobulin biosynthesis and the ability to concentrate the iodide.